Cyclosporin antibiotics are prepared by fermentation. For the first time Swiss patent No. 589,716 reported the isolation of cyclosporins A and B obtained by cultivating the fungus strain Cylindrocarpon lucidum Booth 5760. 25 cyclosporins have been known so far, which are designated by letters A and Z, as reported in Helv. Chim. Acta 70, 13 (1987).
The different components, the chemical and physical properties of which, which are very close to each other, are isolated from the fermentation liquor by extraction. The components are separated from the thus obtained crude cyclosporin mixture by a multistep chromatographic preparative method. Although cyclosporin A which has a selective immunosuppressive activity is the most valuable among the components of the cyclosporin complex, the other components are generally also isolated and purified.
According to a method described in U.S. Pat. No. 4,117,118 the cyclosporin mixture is transferred first to a Sephadex LH20 column and eluted with methanol, then it is eluted successively in an alumina column with a mixture of toluene and ethyl acetate (15%), and in a silica gel column with a mixture of chloroform and methanol (2%). Despite of the repeated chromatography the resulting product is not pure, but it is a mixture of cyclosporins A and B.
According to a process disclosed in U.S. Pat. No. 4,215,199 a mixture of mainly cyclosporins A and B obtained from a fermentation liquor by repeated extraction with ethylene chloride subsequent to mechanical treatment in methanol, is first defatted with a 98:2 v/v mixture of chloroform and methanol on a silica gel column. The eluate is then evaporated to dryness. The residue is dissolved in methanol and is subjected to chromatography in a Sephadex LH20 column using methanol as eluent. The eluate is dissolved in a 98:2 v/v mixture of chloroform and methanol and is again subjected to chromatography in a silica gel column. Cyclosporin A appears first in the eluate, followed by cyclosporin B. The pure components are obtained by evaporating the eluates.
A method is disclosed in Helv. Chim. Acta 59, (4), p. 1075-92 for the separation of mixtures containing cyclosporins A and B. According to this method a fermentation liquor containing cyclosporins A and C is mixed with n-butyl acetate and is mechanically treated in a Westfalia separator. The organic phase is evaporated after that treatment, and the crude cyclosporin mixture is defatted with methanol and petroleum ether. After evaporation the residue is dissolved in chloroform and is subjected to chromatography by gradient elution with 98:5:1.5 v/v and 97:3 v/v mixtures of chloroform and methanol as eluent. Cyclosporin A, appears in the first eluent, cyclosporin B can be found in the second one. The pure crystalline produced is obtained by further chromatography. Fraction A is dissolved in methanol and is subjected to chromatography in a Sephadex LH-20 column by using methanol as eluent. The peak fractions are evaporated, dissolved in toluene and subjected to chromatography in a column packed with aluminium oxide, using toluene as eluent, in the presence of an increasing concentration of acetic ester. The fractions are then evaporated and are treated with activated carbon in an alcohol solution to obtain the crystalline product. The crude fraction C is dissolved in methanol and is eluted with methanol in a Sephadex-LH 20 packed column. The peak fractions are dissolved in diethylether and the cyclosporin C is separated by chromatography in a column packed with silica gel, using a 98:2 v/v mixture of chloroform and ethanol as eluent.
The foregoing chromatographic methods for the separation of cyclosporin mixtures are rather lengthy and expensive. The repeated liquid extraction and evaporation steps and the chromatographic operations that follow render these processes uneconomical and unsuitable for industrial scale application.